The eukaryotic nucleus provides a unique environment to separate DNA replication, transcription, and RNA processing events from protein synthesis in the cytoplasm. Communication between the nucleus and the cytoplasm occurs through massive nuclear pore complexes (NPCs) that span the nuclear envelope (NE). Protein or RNA transport in either direction is signal-dependent and requires interaction with an extended family of importin β-related receptors to chaperone the traffic (Stewart, M. L., Baker, R. P., Bayless, R., Clayton, L., Or ant, R. P., Littlewood, T & Matsuura, Y. (2001) FEBS Lett. 498, 145-149). Cargo association with these receptors is regulated by the type of guanine nucleotide (GTP or GDP) bound to the small GTPase, Ran (Gorlich, D. & Kutay, U. (1999) Annu. Rev. Cell Dev. Biol. 15, 607-660; Adam, S. A., Marr, R. S. & Gerace, L. (1990) J. Cell Biol. 111, 807-816). Cytoplasmic Ran is GDP-bound, whereas the GTP form predominates in the nucleus. There is a steep concentration gradient of the respective pools across the NPC. During interphase of the cell cycle, the gradient is maintained by Ran-associated factors separated by the NE (Gorlich, D. & Kutay, U. (1999) Annu. Rev. Cell Dev. Biol. 15, 607-660). Guanine nucleotide exchange factor RCC1 is exclusively nuclear and promotes RanGDP/GTP exchange. In the cytoplasm, RanGAP and RanBP1 accelerate the intrinsic slow rate of RanGTP hydrolysis, cycling the complex back to the GDP form (Bischoff, F. R., Klebe, C., Kretschmer, J., Wittinghofer, A. & Ponstingl, H. (1994) Proc. Natl. Acad. Sci. USA 91, 2587-2591). Cytoplasmic RanGDP has a low affinity for importin β, allowing the free form of the receptor to bind cargo and traffic through the NPC (Weis, K., Ryder, U. & Lamond, A. I. (1996) EMBO J. 15, 1818-1825). Inside the nucleus, higher-affinity RanGTP releases the cargo, then escorts the receptor back to the cytoplasm for another cycle (Gorlich, D., Pante, N., Kutay, U., Aebi, U. & Bischoff, F. R. (1996) EMBO J. 15, 5584-55 94). If GTP hydrolysis is inhibited, importin β remains bound to RanGTP and unavailable for active protein import. Without import, export of mRNA and ribosomal subunits are also inhibited because these processes require continuous nuclear reuptake of proteins that comprise export-competent ribonucleoproteins (RNPs) (Gorlich, D. & Kutay, U. (1999) Annu. Rev. Cell Dev. Biol. 15, 607-660). Therefore, if the Ran gradient fails, only proteins small enough to diffuse through the NPC (<40-60 kDa) can exchange.
The metabolic bottleneck posed by nuclear trafficking makes the NPC and associated processes vulnerable to attack by viruses intent on impeding signal transduction to the nucleus. NPC abrogation can prevent up-regulation of antiviral genes and the export of detrimental cellular mRNAs or enhance the redistribution of nuclear proteins required for viral replication (Belov, G. A., Lidsky, P. V., Mitkitas, O. V., Egger, D., Lukyanov, K. A., Bienz, K. & Agol, V. (2004) J. Virol 78, 10166-10177.). The family of RNA Picornavirus includes a variety of pathogenic agents. Among the better known members are poliovirus, rhinovirus, and foot-and-mouth disease virus. Others include the Coxsackie viruses, hepatitis A, swine vesicular disease, and cardioviruses like Mengo virus, encephalomyocarditis virus (EMCV), and Theiler's murine encephalomyelitis virus (Palmenberg, A. C. (1987) in Positive Strand RNA Viruses, eds. Brinton, M. A. & Rueckert, R. R. (Liss, New York), pp. 25-34.). These positive-sense RNA genomes direct a life cycle that is predominantly cytoplasmic and, indeed, can be recapitulated in a test tube in the absence of nuclei (Molla, A., Paul, A V. & Wimmer, E (1991) Science 254, 1647-1651.). During infection, however, Picornaviruses are adept at subverting innate cellular immunity traps, crippling the capacity of the cell to mount a defense. Within 2-3 h, infection brings to a halt all cellular mRNA transcription, cap-dependent mRNA translation, antiviral signal transduction, and active protein/RNA exchange between the nucleus and cytoplasm. The shutoff is profound. The viruses replicate with fecundity, and the cell dies before it ever triggers an alarm. Among the molecular processes involved in shutoff, poliovirus and rhinovirus encode a protease, 2Apro, which attacks nucleoporins within the NPC (Belov, G. A., Lidsky, P. V., Mitkitas, O. V., Egger, D., Lukyanov, K. A., Bienz, K. & Agol, V. (2004) J. Virol 78, 10166-10177; Gustin, K. E. & Sarnow, P. (2002) J. Virol. 76, 8787-8796; Gustin, K. E. & Sarnow, P. (2001) EMBO J. 20, 240-249). When visualized by electron microscopy, a “bar-like” structure spanning normal NPC channel is found to be missing (Belov, G. A., Lidsky, P. V., Mitkitas, O. V., Egger, D., Lukyanov, K. A., Bienz, K. & Agol, V. (2004) J. Virol 78, 10166-10177), and its absence correlates with an onset of unregulated efflux of small proteins from the nucleus into the cytoplasm.
Cardioviruses like EMCV also abrogate nucleocytoplasmic trafficking, but their genomes lack a 2A protease and instead encode another protein, the Leader (L), at the amino terminus of the viral polyprotein (FIG. 1A). The L protein is 67 aa long, with a novel CHCC zinc-finger motif, a highly acidic carboxyl domain (protein pI: 3.8), and no known homologs (FIG. 1). EMCV with L deletions are viable but have attenuated growth phenotypes (Dvorak, C. M. T., Hall, D. J., Hill, M, Riddle, M., Pranter, A., Dillman, J., Deibel, M. & Palmenberg, A. C. (2001) Virology 290, 261-271). They are inefficient at shutting off host protein synthesis (Zoll, J., Galama, J. M.D., van Kuppeveld, F. J. M. & Melchers, W. J. G. (1996) J. Virol. 70, 4948-4958), and they stimulate an increased antiviral IFN activity (Zoll, J., Melchers, W. J., Galama, J. M. & van Kuppeveld, F. J. (2002) J. Virol. 76, 9664-9672). For Theiler's murine encephalomyelitis virus the related L likewise affects nuclear trafficking. This slightly larger L (76 aa) stimulates IFN transcriptional activator IRF-3, normally a cytoplasmic entity, to redistribute aberrantly between the nucleus and cytoplasm. Simultaneously, polypyrimidine tract-binding protein, a nuclear component of premRNA splicing complexes, redistributes to the cytoplasm and is usurped into viral replication machinery. As a result, viral replication is enhanced by the cytoplasmic availability of polypyrimidine tract binding protein, and the cell is unable to mount a viable IFN-dependent antiviral response to the infection. The antitrafficking activities of EMCV and Theiler's murine encephalomyelitis virus are compromised if either virus harbors a mutation in the zinc-finger region of the L protein (Delhaye, S., van Pesch, V. & Michiels, T (2004) J. Virol. 78, 4357-4362). It has been proposed that the cardiovirus L, like the poliovirus/rhinovirus 2Apro, disrupts the integrity of the NPC, leading to the leakage of nuclear proteins by passive diffusion (Lidsky, P. L., Hato, S., Bardina, M. V., Aminev, A. G., Palmenberg, A. C., Sheval, E. V., Polyakov, V. Y., van Kuppeveld, F. J. & Agol, V. (2006) J. Virol. 80, 2705-2717). However, it is difficult to envision how a small protein, lacking any known enzymatic activity, could effectively attack the massive NPC with the scant copy number presented to an infected cell.
In the work presented below, we hypothesized that an alternative target might be the nuclear transport system composed of Ran-GTPase or the cofactors required for maintaining the RanGTP gradient. We now describe experiments showing that the EMCV L binds directly to Ran and disrupts nucleocytoplasmic trafficking.